Non-ramified culture growing apparatus

ABSTRACT

A non-ramified culture growing apparatus wherein an inner domed surface of a glass bell-jar provides a culture draining surface, and culture is circulated by a fluid operated, diaphragm pump from a lower portion of the bell-jar along a pipe to be sprayed on to the draining surface by a nozzle.

United States Patent [in Dawson et a1.

Jan. 30, 1973 NON-RAMIFIED CULTURE GROWING APPARATUS Inventors: PeterS.- S. Dawson; Wolfgang G. W. Kurz; Moiiat Anderson; Arthur E.

York, all of Saskatoon, Saskatchewan, Canada Assignee: Canadian Patentsand Development Limited, Ottawa, Ontario, Canada Filed: March 8, 1971App1.No.: 121,694

Foreign Application Priority Data May 8, 1970 Canada ..082,268

U.S. C1. ..195/l43,195/109,195/127, 195/139 Int. Cl. ..Cl2b 1/00 Fieldof Search ..195/143, 109, 127, 139; 222/190 [56] References Cited UNITEDSTATES PATENTS 3,201,327 8/1965 Beck ..l95/143 X Primary ExaminerA.Louis Monacell Assistant ExaminerWi1liam Andrew Simons Attorney-James R.Hughes [57] ABSTRACT A non-ramified culture growing apparatus wherein aninner domed surface of a glass bell-jar provides a culture drainingsurface, and culture is circulated by a fluid operated, diaphragm pumpfrom a lower portion of the bell-jar along a pipe to be sprayed on tothe draining surface by a nozzle.

9 Claims, 5 Drawing Figures PATENTEDJAN 30 I973 3,713,988

sum 2 or '3 STAGE I IN law Mar-F47 Armin Arm/z an 4; A? z 1' INON-RAMIFIED CULTURE GRGWING APPARATUS This invention relates to anon-ramified culture growing apparatus.

Known types of culture growing apparatus use circulation pumps whichhave impeller blades and these pumps cannot be used for growing culturesof organisms which are sensitive to the shearing effects of the impellerblades.

Itiis an object of the invention to provide a culture growing apparatuswhich has a circulationpump having no impeller blades, and which thusfacilitates the use of the apparatus for growing cultures of organismssensi-' tive to the shear stresses of pump impellers. v

The culture growingapparatus of the invention, however, has not beenfound suitable for growing cultures of filamentous organisms producingramified growths.

According to the invention there is provided a nonramified culturegrowing apparatus, comprising a vessel sealed from the surroundingatmosphere and having a culture draining surface in an upper portionthereof, conveying means for conveying culture from a lower portion ofsaid vessel to said upper portion thereof, dispersing means fordispersing said conveyed culture as a film for draining down saiddraining surface to said lower portion, a fluid operated diaphragm pumpfor pumping culture along said conveying means, and a non-return valveconnected to the culture inlet of said pump for causing culture to flowin the pumpin direction.

In the accompanying drawings which illustrate, by

way of example, embodiments of the invention, v FIG. 1 is a partlysectioned side view of aculture vessel of a culture growing apparatus,

, FIG. 2 is an enlarged sectional side view of a part of the culturevessel shown in FIG. 1, revealing the diaphragm pump,

FIG. 3 is a partly sectioned outline of the apparatus shown in FIGS. 1and 2, connected to medium supply and culture receiving vessels,

reinforced neoprene flap-valve 17 is connected by a .bolt 19 to the baseplate 3 to overlap culture inlets 18 of thepump 14 for causing cultureto flow in the pum ping direction. A source of pumping fluid, in thisinstance a pressurized air from a central supply 20, containing air at12 to 15 p.s.i., is connected to an inlet pipe 21 to the diaphragm pump14 via a needle valve The rubber gasket 5 is clamped between the glassrim 4 and the base plate 3 by a brass retaining ring 24 through whichbolts 25 provided with washers 26 pass and are screwed into the baseplate 3. The base plate 3 is provided with four legs 28. A culture inletfeed pipe 29 and an air inlet pipe 30 to, and an air outlet pipe 31 fromthe bell-jar leach extend through and are sealed to the base plate'3.The air inlet pipe 30, which has a valve 33, and air outlet pipe 31,which has a valve 35, are each fitted with a glass fiber filter (notshown).

The pipe 10 has a number of small stainless steel tubes 32"(FIG. 1)joined to it over perforations therein. The tubes 32 each extend alongorbital-like curved paths from the pipe 10 towards an open end of thattube 32.

The diaphragm 15 (FIG. 2) seals a culture cavity 34 in the base plate 3from a annular, air cavity 36 in the body 16. The cavity 34 is open tothe base of the pipe 10 and is sealed from the inlets 18 by the flapvalve 17. The cavity 36 has an air inlet 21 and air outlets 38surrounded by an annular rim 41. A stainless steel culture outlet pipe39 extends through the base plate 3 to one FIG. 4 is a diagrammatic viewof a multistage chemostat assembly, and

FIG. 5 is a diagrammatic view of an apparatus for phased or synchronousculture growth.

In FIGS. 1 and 2 there. is shown a vessel in the form of a heatresisting soda-alumina-borosilicate, plain top glass bell-jar 1 which issealed by means of a ground glass rim 4 from the surrounding atmosphereto a stainless steel base plate 3 by a rubber gasket 5. The insidesurface 7 of the domed upper portion 8 of the bell-jar forms a culturedraining surface in the upper portion of the bell-jar l. A culturedispersing pipe 10 (shown partly sectioned) in the bell-jar'1 forms aconveying means for conveying culture from a lower portion 11 of thebell-jar 1. Dispersing means in the form of a polytetrafluoroethylenenozzle 13 is provided for dispersing culture down the surface 7 to thelower portion 11, after it has been conveyed along the pipe 10. Thenozzle 13 is a piece of tubing which has been forced over the end of thepipe 10 and is spaced onesixteenth inch from a central portion of thesurface 7 to form an umbrella-shaped flow of culture.

A fluid operated diaphragm pump generally designated 14 (FIG. 2) isformed from a portion of the base plate 3, a reinforced neoprenediaphragm 15, and

side of the brass body 16'. The pipe 39 extends into the bell-jar 1 totrap culture 40 in the lower portion thereof.

In the embodiments shown in FIG. 3 a diagrammatic side view of theapparatus of FIG. 1 is shown connected to a vessel 42 containing anon-ramified culture grow ing medium 44, and a vessel 45 for receiving aharvest 46 of non-ramified culture from the culture 40.

The medium inlet feed pipe 29 is connected to the vessel 42 by tubing 48containing a clamp 49 and flow meter 50. A pump 52 is provided forpumping the medium 44 from the vessel 42 through the tubing 48 into thebell-jar 1 through the'feed pipe 29. The vessel 42 is a glass jar fittedwith a bung 55 through which the tubing 48 extends. A vent tube 56 alsoextends through the bung 55 and is fitted with an air filter 57.

The'vessel 45 is connected by tubing 59 to the culture outlet pipe 39.The vessel 45 is a glass jar fitted with a bung 60 through which thetubing 59 extends. A vent tube 61 also extends through the bung 60 andis fitted with an air filter 62.

In operation the apparatus is assembled as shown in FIG. 1 without theculture 40 therein, and not connected to the additional apparatus shownin FIG. 3. It should be noted that the air inlet pipe 21 is notconnected to the air supply 20 (FIG. 2) or needle valve 22.

The medium inlet feed pipe 29 is plugged by a plug (not shown) and aone-way stopcock (not shown) is attached to the air inlet pipe 30. Theapparatus is then sterilized in an autoclave (not shown).

After being cooled the apparatus is connected to the additionalapparatus shownin FIG. 3 and then inoculated with culture 40 underaseptic conditions. Fresh medium is provided by activating the pump 52.The apparatus is then connected to the air supply 20 and the needlevalve 22.

With the valve 35 closed the valve 33 is opened to admit air to theinterior of the bell-jar 1. Since air cannot escape from the bell-jar 1the air pressure inside it increases, and this is allowed to continueuntil a pressure of 3 p.s.i. has been attained therein. This airpressure forces culture 40 through the flap valve 17 and into theculture cavity 34. The culture 40 moving into the culture cavity 34causes the diaphragm 15 to be pressed against the annular rim 41 andseal the outlets 38. The needle valve 22 is then opened permanently, thevalve 17 is closed, and the air pressure within the air cavity 36 isthen allowed to build up to pressurize the culture 40 within the culturecavity 34, through the diaphragm 15, and close the flap valve 17. As theair pressure within the air cavity 36 increases further the culture 40trapped within the culture cavity 34 is forced by the diaphragm 15 intothe culture dispersing pipe 10. The culture 40 travels up the pipe andis dispersed on to the surface 7 to form a thin, umbrellashaped flowingfilm of culture draining down the surface 7, as illustrated by thearrows X in FIG. 1.

As the culture 40 is discharged from the culture cavity 34 the diaphragmuncovers the air outlets 38 and allow air to escape through them fromthe air cavity 36. At this point the diaphragm collapses allowing freshculture 40 to enter the culture cavity 34 under the pressure existing inthe glass bell-jar 1, and the cycle is repeated once more. Thus thepressure in the glass belljar 1 repeatedly forces the culture 40 intothe culture cavity 34 to seat the diaphragm 15 on to the rim 41, andeach time this occurs the air pressure in the air cavity 36 increases toclose the flap valve 17 and force culture 40 up through the pipe 10until the air pressure in the air cavity 36 drops sufficiently for theflap valve 17 to open once more and the diaphragm 15 seals on the rim 41once more. It should be noted that a small portion of the culture 40which is passed to the pipe 10 flows out of the tube 32 to gentlyagitate the culture 40 in the lower portion of the glass bell-jar 40.

The apparatus may be used for batch, single or multistage chemostat, orphased culture. In the arrangement shown in FIG. 3 the apparatus isshown used for single stage chemostat operation.

With the apparatus functioning as described above and connected to theadditional apparatus shown in FIG. 3, a sterile supply medium 44 ispumped continuously from vessel 42 by pump 52 into the glass bell-jar 1through feed pipe 29. The pump 52 is a known piston type metering pumpmade of stainless steel, and the rate of flow of supply medium 44through the pump can be adjusted by a micrometer adjustment (not shown)of the piston stroke (not shown) until the desired rate of flow isindicated by the flowmeter 50. A constant volume of culture 40 isestablished in the glass bell-jar 1 by the culture outlet pipe 39 intowhich excess culture 40 overflows and passes to the vessel 45. It shouldbe noted that on the air filter 62 a valve similar to valve 35 (FIG. 1)is attached to retain the air pressure at around 3 p.s.i. in the glassbell-jar 1.

In order to use the apparatus shown in FIG. 3 for batch production thepump 52 is operated intermittently to replenish the medium 40 within theglass belljar 1.

In FIG. 4 there is shown a diagrammatic view of a multistage chemostatassembly incorporating three culture growing apparatus of the type shownin FIGS. 1 and 2. Similar parts in FIG. 4 to those shown in FIGS. 1 to 3are designated by the same reference numerals, and the previousdescription is relied upon to describe them. Not all of the apparatusshown in FIGS. 1 and 2 is shown in FIG. 4, but it will be appreciatedthat even though it is not shown it is in fact intended to be present.

It should be noted that the culture outlet pipes 39 from the first twoglass bell-jars l areeach connected to culture inlet feed pipe 29a tothe next glass bell-jar 1, whilst the medium inlet feed pipe 29 of thefirst glass bell-jar 1 is connected to the vessel 42. Thus each assemblysimilar to that shown in FIGS. 1 and 2 provides fermentation stagebetween the vessel 42 and the vessel 45.

The operation of the apparatus shown in FIG. 4 is similar to theoperation of the apparatus shown in FIGS. 1 to 3 except that the culture40 is pumped from one glass bell-jar l to another until it is collectedin the vessel 45.

The variations that can be made in the apparatus are numerous. Forexample the successive stages can be made of different capacities, thevolume of the culture 40 can be changed by altering the height of theoutlet pipes 39 in their respective glass bell-jar. A straight throughflow of culture may be used as illustrated or additions may be made atany of the stages of supplements such as, for example, precursors,specific nutrients, or reagents.

Air supplied to the first stage through the air inlet pipe 30subsequently passes out with the culture 40 through outlet pipe 39 andhelps to maintain the flow of culture 40 from one glass bell-jar 1 tothe next one. Additional air may be supplied to any one or all of thesubsequent stages as required through respective air inlet pipes 30(shown chain dotted) in each stage.

It will be appreciated that more or less than the three stages shown inFIG. 4 may be used if desired.

In FIG. 5 there is shown a diagrammatic view of an apparatus used forphased or synchronous culture growth, and incorporating two of theassemblies shown in FIGS. 1 and 2. In FIG. 5 similar parts to thoseshown in FIGS. 1 and 2 are designated by the same reference numerals,and the previous description is relied upon to describe them. Whilst allof the apparatus shown in FIGS. 1 and 2 is not illustrated in FIG. 5, itwill be understood that it is all intended to be included in theprocessing unit assembly 68.

In FIG. 5 there is shown an assembly 66 and an assembly 68. The assembly68 is similar to the apparatus shown in FIGS. 1 and 2 except that aculture outlet pipe 67 is provided which does not extend into thebell-jar 1 to trap culture and it is controlled by an exit clamp 89. Theassembly 66 is a phasing unit and includes only the apparatus shown fromthat illustrated in FIGS. 1 and 2 and the assembly 68 is a processingunit. A medium supply vessel 69 contains supply medium 70. The vessel 69has an air filter 72 and a supply pipe 74. An outlet pipe 76 from thevessel 69 is connected to a solenoid valve 77, which in turn isconnected to a dosing vessel 78.'An outlet 80 from the vessel 78 isconnected to a peristaltic pump 82, and an outlet 84 from theperistaltic pump 82 is connected to the inlet pipe 29 of the asfrom theassembly 68 delivers culture to a sampler 88 t or to the vessel 45.

The-dosing vessel 78 has an air filter 89, and two electrodes 90 and 91which are of different lengths and both connected to a relay 93 (shownchain-dotted).

The relay 93 is connected to the solenoid valve 77 and to a timer 94(shown chain-dotted). The timer 94 is connected to the peristaltic pumps82 and'86. Clamps 90 and 92 are provided in addition to the clamp 89. Ifdesired clamps 91 and96 may replace clamps 90 and 92 respectively.

in operation the assembly 68 operates as previously described. The relay93 actuates the solenoid valve 77 therein. After a short interval thetimer 94 causes'theperistaltic pump 86 to remove one half of the mixedculture 40 in the assembly 66 and pass it to the vessel 45, or to theassembly 68 where the culture grows under conditions identical to thoseexisting in the asln general the apparatus of the invention has beenfound to possess several additional advantages over known types offermenting apparatus, such as, for example, the production of a veryhomogeneous culture,

the relatively fast cycle of agitation (for example 100 ml per second)of the culture prevents any formation of wall growth on the innersurface of the glass bell-jar 1, and permits representative harvestingof samples of the culture.

The apparatus of the invention has been used having a working capacityof 1,000 ml, but this working capacity may be changed with the apparatusused to any desired capacity of, for example, 500 to 1,5000 ml byadjusting the level of the outlet pipe 39 in the glass bell-jar. Withthis apparatus the culture draining surface 7 was 1,100 cm and the filmof culture draining down it was 0.33 mm thick. Approximately 40 ml ofthe 1,000 mlof-culture was retained in the draining film. As alreadystated the culture 40 was circulated at the rate of 100 ml per second,which gave a circulation turnover time of ten seconds, and a transittime in the draining of the film of culture on the surface 7 of 0.38seconds. The conditions of aeration were found to be similar to thoseobtained in the so-called cyclone column apparatus, and were foundcomparable with those observed in known types of stirred vessels.

Like the so-called cyclone column fermentation ap- 'paratus theapparatus of the invention does not promote foam formation and sopermits the use of substrates having high foaming potential, such asmolasses sembly 66. Manual manipulation of clamps 90 and 91 I allowdischarge of culture 40 from assembly 66 to harvest vessel or assembly68. Clamp 89 retains culture 40 in assembly 68. The opening of clamps 89and 92 or 96 discharges culture 40 from assembly 68 to either sampler 88or harvest vessel 45. The solenoid valve 77 once again is then actuatedby the timer 94 and relay 93 to refill the dosing vessel 78 so that thecycle may be repeated once again. The assembly 68 operates in the samemanner as the apparatus shown in FIGS. 1 and 2 and delivers culture tothe vessel 45. The sampler 88is provided for sampling culture deliveredfrom the assembly 68.

The apparatusof the-invention has'been found to be a relatively simpleand inexpensive apparatus for use, for example, in conducting. certainexploratory studies of multistage, continuous culture growth inlaboratories. Whilst it has been found that the apparatus of theinvention lacks the sophisticated performance of the so-called cyclonecolumn culture growing apparatus, and the possibility of cultivating.mycelial type (ram ified) organisms, it retains the other advantages ofthe cyclone system and furthermore-permits the cultivations ofthosesorganismsand tissues which are sensia tive to the shearing effectsof the pumps used in known apparatus.

Apparatus of the. invention may be used, for example, for chemostatstudies with yeast and bacteria, and for growing-plant cells and algae.The high aeration of the culture, and the large. ratio of surface areato culture volume obtained by the umbrella-shaped flowing film ofculture, are :experimental advantages of the apparatus of the invention,especially for the photosynthetic organisms.

or peptones, without the addition of antifoam agents.

The apparatus of the invention may be used in a thermostat ortemperature controlled room, and these conditions have been foundadequate for ensuring that the culture is maintained under rigoroustemperature control. The circulation of culture 40 produced by thediaphragm l5 eliminates any complications from being introduced by thejoule effect heating of the culture 40 by conventional mechanical mixingsystems. This in turn eliminates the need for a cooling system andretains the simplicity of the apparatus of the invention. This leads toadvantages of the apparatus of the invention, when compared withconventional apparatus, in that the apparatus is relatively inexpensive,simple in operationand control.

Organisms with which the apparatus of the invention has been used are:

Bacillus subtilis Azotobacter vinelandii Candida utilis Saccharomycesrouxii, and

Plant tissue cells such as Vicia, Rosaand Reseda.

We claim:

1. Non-ramified culture growing apparatus, comprising a vessel sealedfrom the surrounding atmosphere a'ndhaving a culture draining surface inan upper portion thereof, conveying means for conveying culture fromalower portion of said vessel to said upper portion thereof, dispersingmeansfor dispersing said conveyed culture as a film for draining downsaid draining surface to said lower portion, a fluid operated diaphragmpump for pumping culture along said conveying means, and a non-returnvalve connected to the culture inlet of said pumpfor causing culture toflow in the pumping direction.

2. Apparatus according to claim 1, which includes a plurality of smalltubes along orbital like curved paths from said conveying means forgently agitating culture when it is disposed in a lower portion of saidvessel.

3. Apparatus according to claim 1, wherein said fluid operated diaphragmpump comprises a base plate sealing a lower end of said vessel andhaving a culture cavity with said culture inlet thereto from theinterior of said vessel, a diaphragm sealing the lower side of saidculture cavity, a body portion having an air cavity with an air inletand an air outlet thereto, said diaphragm being disposed between saidbase plate and said base plate to seal said culture cavity from said aircavity, and displaceable by air pressure in said air-cavity to forceculture out of said culture cavity.

4. Apparatus according to claim 3, which includes a continuous lip insaid air cavity and around said air outlet, such that pressure fromculture within said cavity will cause said diaphragm to press againstsaid lip and seal said air outlet.

5. Apparatus according to claim 1, which includes a culture outlet pipeextending upwardly into said vessel to form an overflow pipe for culturewhen it is disposed in the lower portion of said vessel.

6. Apparatus according to claim 5, wherein the height of said cultureoutlet pipe within said vessel is adjustable for adjusting the volume ofculture when it is disposed in the vessel.

7. Apparatus according to claim 6, which includes a dosing meansconnected to receive culture from said supply vessel, a phasing unitconnected to receive a predetermined volume of culture from said dosingmeans, and deliver it to said vessel having said draining surface, andcontrol means for delivering from said dosing means, when the apparatusis in operation, said predetermined volume of culture to said phasingunit and, after a predetermined time interval, removing a predeterminedquantity of said culture in said vessel having said draining surface anddelivering it to said harvest vessel.

8. Apparatus according to claim 6, which includes a culture mediumsupply vessel, means for pumping culture from said supply vessel to saidvessel having said draining surface, and a culture harvest vesselconnected to receive culture from said culture outlet pipe.

9. Apparatus according to claim 8, wherein said vessel having saiddraining surface is one of a plurality of such vessels, each forming afermentation stage in series flow between said supply vessel and saidharvest vessel.

1. Non-ramified culture growing apparatus, comprising a vessel sealedfrom the surrounding atmosphere and having a culture draining surface inan upper portion thereof, conveying means for conveying culture from alower portion of said vessel to said upper portion thereof, dispersingmeans for dispersing said conveyed culture as a film for draining downsaid draining surface to said lower portion, a fluid operated diaphragmpump for pumping culture along said conveying means, and a non-returnvalve connected to the culture inlet of said pump for causing culture toflow in the pumping direction.
 2. Apparatus according to claim 1, whichincludes a plurality of small tubes along orbital like curved paths fromsaid conveying means for gently agitating culture when it is disposed ina lower portion of said vessel.
 3. Apparatus according to claim 1,wherein said fluid operated diaphragm pump comprises a base platesealing a lower end of said vessel and having a culture cavity with saidculture inlet thereto from the interior of said vessel, a diaphragmsealing the lower side of said culture cavity, a body portion having anair cavity with an air inlet and an air outlet thereto, said diaphragmbeing disposed between said base plate and said base plate to seal saidculture cavity from said air cavity, and displaceable by air pressure insaid air-cavity to force culture out of said culture cavity. 4.Apparatus according to claim 3, which includes a continuous lip in saidair cavity and around said air outlet, such that pressure from culturewithin said cavity will cause said diaphragm to press against said lipand seal said air outlet.
 5. Apparatus according to claim 1, whichincludes a culture outlet pipe extending upwardly into said vessel toform an overflow pipe for culture when it is disposed in the lowerportion of said vessel.
 6. Apparatus according to claim 5, wherein theheight of said culture outlet pipe within said vessel is adjustable foradjusting the volume of culture when it is disposed in the vessel. 7.Apparatus according to claim 6, which includes a dosing means connectedto receive culture from said supply vessel, a phasing unit connected toreceive a predetermined volume of culture from said dosing means, anddeliver it to said vessel having said draining surface, and controlmeans for delivering from said dosing means, when the apparatus is inoperation, said predetermined volume of culture to said phasing unitand, after a predetermined time interval, removing a predeterminedquantity of said culture in said vessel having said draining surface anddelivering it to said harvest vessel.
 8. Apparatus according to claim 6,which includes a culture medium supply vessel, means for pumping culturefrom said supply vessel to said vessel having said draining surface, anda culture harvest vessel connected to receive culture from said cultureoutlet pipe.